The present invention relates to a method for adhering and proliferating cell. Particularly, the present invention relates to a method for adhering and proliferating epithelial and hepatic cell. The present invention also relates to a method for culturing epidermal cell to be used in an epidermal cell sheet and an epidermal cell suspension which can be applied to an apellous part such as those with burn, wound, bedsore or skin ulcer for early reconstruction or treatment of such damaged tissue, an epidermal cell sheet and suspension prepared by the culture method, and a method for culturing hepatic cell which is important in analysis of hepatic function.
The present invention also relates to a culture vessel manufactured according to the steps of the methods of the present invention, which can provide improved adhesion to cell and enhanced cell-proliferation. Particularly, the present invention relates to a culture vessel which can provide improved adhesion to epithelial and/or hepatic cell and enhanced epithelial and/or hepatic cell-proliferation. More particularly, the present invention relates to a culture vessel that enables preparation of an epidermal cell sheet and an epidermal cell suspension to be applied to an apellous part such as those with burn, wound, bedsore or skin ulcer for early reconstruction or treatment of such damaged tissue.
Conventionally, two methods have widely been employed for culturing epithelial cell, particularly epidermal cell (or which is called epidermal keratinocyte). One utilizes sterilized 3T3 mouse embryo fibroblast, i.e., viable 3T3 mouse embryo fibroblast from which division and proliferation potencies have been deleted by irradiating, for example, xcex3 ray or by adding an agent such as mitomycin C, as feeder layer (such as the feeder layer culture method described in James G. Rheinwald and Howard Green. Cell 6:331-344. Serial Cultivation of Strains of Human Epidermal Keratinocytes: the Formation of Keratinizing Colonies from Single Cells). The other utilizes serum-free medium such as MCDB153 instead of feeder layer.
However, the conventional feeder layer culture method in which 3T3 mouse embryo fibroblasts are used as a feeder layer involves complicated procedure for preparing the 3T3 mouse embryo fibroblasts immediately before epidermal cells are inoculated, and such feeder layer has a limited life time. During proliferation of epidermal cells other than mouse epidermal cells such as human epidermal cells and preparation of an epidermal cell sheet, those cells may possibly be contaminated with heterogenous cells, 3T3 mouse embryo fibroblasts, and an agent such as mitomycin C, which is added to delete division and proliferation potencies of the 3T3 embryo fibroblasts, may remain.
On the other hand, when fibroblast homogenous to the epidermal cell and/or epidermal cell sheet of interest is used as the feeder cell instead of 3T3 mouse embryo fibroblast (e.g., human fibroblast may be used for preparing a human epidermal cell sheet), there is no possibility of contamination with heterogenous cells. However, their division and proliferation potencies should also be deleted by adding an agent such as mitomycin C that may possibly remain. Human fibroblast may be used, but they provide slower proliferation rate of the epidermal cells when compared to that obtained by using 3T3 mouse embryo fibroblast.
Culture method employing serum-free medium may often provide slower proliferation rate of epidermal cell and require longer period of time for incubation when compared to feeder layer culture methods which employ feeder cells such as 3T3 mouse embryo fibroblast. Further, serum-free medium is likely to suppress the differentiation of epidermal cell, which may cause inability of the epidermal cell to form a multiple-layer, resulting in unsuccessful preparation of an epidermal cell sheet.
Japanese Unexamined Patent Application Publication No. 285781/1987 disclosed a method which employs a feeder layer to culture hepatic cells. This method may also have a possibility of residual agent or contamination with heterogenous cells. Further, this method requires continual subculture of feeder cell to keep the cell ready for use under an optimal condition (subconfluent), making the procedure very complicated.
According to the investigation of various culture conditions for epidermal cell forming an epidermal cell sheet and hepatic cell, a novel method is found, which comprises the steps of inoculating, culturing and then killing fibroblast derived from a mammal and separating the killed fibroblasts from the vessel at least partially to substantially leave a component or components, on the surface of the culture vessel, such as the accumulated extracellular matrix which has been secreted from the culture cells. Thus, this method does not require preparing feeder cells such as fibroblasts nor sterilizing the cells as required in conventional feeder layer culture methods. Further, a culture vessel manufactured according to the steps of the above method, which can provide improved adhesion to cell and enhanced cell-proliferation, may enable to be preserved for long time while keeping its property. Accordingly, the culture vessel does not require daily subculture (i.e., transferring cells on another vessel in order to prevent overpopulation of the cells in the culture vessel) of fibroblasts to be used as the feeder cells. Preservation of a large stock of the culture vessels can be preserved in a cold and dark place. Further, the culture vessel which can provide improved adhesion to cell and enhanced cell-proliferation when compared to one manufactured by conventional methods can be obtained. On the basis of these findings, the present invention has been completed.
Accordingly, one object of the present invention is to provide a method for adhering and proliferating cell comprising the steps of inoculating, culturing and then killing fibroblast derived from a mammal, which can provide improved cell adhesion and proliferation potencies when compared to those provided by conventional feeder layer culture method, without preparing feeder cells such as fibroblasts nor sterilizing such cells as required in conventional feeder layer culture methods, and thus can provide an epidermal cell sheet, an epidermal cell suspension or hepatic cells while avoiding contamination with heterogenous cells. Another object of the present invention is to provide the followings: a method for culturing epidermal cell to be used in an epidermal cell sheet and an epidermal cell suspension which can be applied to an apellous part such as those with burn, wound, bedsore or skin ulcer for early reconstruction or treatment of the damaged tissue; an epidermal cell sheet and an epidermal cell suspension prepared by the culture method; and a method for culturing hepatic cells which is important in analysis of hepatic function.
Still another object of the present invention is to provide a culture vessel which can provide improved adhesion to cell and enhanced cell-proliferation according to the steps of the above method. Particularly, one object of the present invention is to provide a culture vessel which can provide improved adhesion to cell and enhanced cell-proliferation, which are manufactured by culturing and killing fibroblasts derived from a mammal (particularly 3T3 mouse embryo fibroblast) by, for example, freezing and/or drying in a culture vessel and separating the killed fibroblasts from the vessel at least partially to substantially leave a component or components such as the accumulated extracellular matrix which has been secreted from the culture cells to remain on the surface of the culture vessel, i.e., to leave component(s) necessary for cell adhesion and proliferation to remain on the surface of the culture vessel.
In summary, the present invention relates to a method for adhering and proliferating cell which comprises the steps of inoculating, culturing and then killing fibroblast derived from a mammal.
In the method, killed fibroblast may be preferably separated from the vessel partially, and more preferably entirely.
In the method, fibroblast may be killed by freezing, drying and/or irradiating electromagnetic radiation or by repeating one treatment selected from the group consisting of freezing, drying and irradiating electromagnetic radiation. Fibroblast may also be killed by a combination of at least two treatments selected from the group consisting of freezing, drying and irradiating electromagnetic radiation. In the method, electromagnetic radiation may be at least one selected from the group consisting of xcex2 ray, xcex3 ray, X-ray, electron beam and UV ray.
In the method, 3T3 mouse embryo fibroblast may be used as fibroblast derived from a mammal.
In the method, epithelial or hepatic cell may be used as the cell which adheres and proliferates. Epidermal cell is preferable as epithelial cell.
Further, the present invention relates to epidermal cell which has been cultured using the method. The present invention also relates to an epidermal cell sheet or suspension prepared from the epidermal cell.
The present invention also relates to a culture vessel manufactured according to the steps of the method, which can provide improved adhesion to epithelial and/or hepatic cell and enhanced epithelial and/or hepatic cell-proliferation.
In the culture vessel, killed fibroblast may be preferably separated at least partially, or entirely.
Preferably, the culture vessel can be preserved by freezing and/or drying.
For the culture vessel, 3T3 mouse embryo fibroblast is used as fibroblast derived from a mammal preferably.
For the culture vessel, epidermal keratinocyte may be used preferably as the epithelial cell.
The material of the culture vessel may be glass, synthetic polymer or biopolymer. Further, the culture vessel may have any shape such as flask, petri dish, roller bottle, tray, well plate, beads, film, sheet or sponge.
The culture vessel made of glass or synthetic polymer may have any shape such as flask, petri dish, roller bottle, tray, well plate, beads, film, sheet or sponge.
Preferably, the culture vessel made of biopolymer may have any shape such as sheet, film, sponge or beads.
The culture vessel may be used to prepare an epidermal cell sheet.
The culture vessel may be used to prepare an epidermal cell suspension.